Solid phase peptide synthesis

ABSTRACT

An improved method of deprotection in solid phase peptide synthesis is disclosed. In particular the deprotecting composition is added in high concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated acid from the preceding coupling cycle, and without any draining step between the coupling step of the previous cycle and the addition of the deprotection composition for the successive cycle. Thereafter, the ambient pressure in the vessel is reduced with a vacuum pull to remove the deprotecting composition without any draining step and without otherwise adversely affecting the remaining materials in the vessel or causing problems in subsequent steps in the SPPS cycle.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 15/299,931 filed Oct. 21,2016 and claims benefit of Serial No. PCT/US16/58181 filed Oct. 21, 2016and Ser. No. 62/245,484 filed Oct. 23, 2015. This application is relatedto co-pending Ser. No. 15/490,090 filed Apr. 18, 2017 as acontinuation-in-part of Ser. No. 15/299,931 and PCT/US17/28254 filedApr. 19, 2017 which claim benefit of Ser. No. 62/383,397 filed Sep. 3,2016.

BACKGROUND

The present invention relates to improvements in the solid phasesynthesis of peptides (“SPPS”).

Peptides are linked chains of amino acids which in turn are the basicbuilding blocks for most living organisms. Peptides are also theprecursors of proteins; i.e., long complex chains of amino acids.Peptides and proteins are fundamental to human and animal life, and theydrive, affect, or control a wide variety of natural processes.

As just one example, peptides have been recently identified that can“keyhole” tumor specific mutations in certain cancers and thus act astumor specific vaccines (e.g., SAMPSON, J H ET AL . An epidermal growthfactor receptor variant III-targeted vaccine is safe and immunogenic inpatients with glioblastoma multiforme. Mol. Cancer Ther. 2009; 8:2773-2779; LI G, SIDDHARTHA M, WONG A J. The epidermal growth factorvariant III peptide vaccine for treatment of malignant gliomas.Neurosurg. Clin. N. Am. 2010; 21: 87-93; LI G, WONG A J. EGF receptorvariant III as a target antigen for tumor immunotherapy. Expert Rev.Vaccines 2008; 7: 977-985).

As a result, the study of peptides and proteins and the capability tosynthesize peptides and proteins are of significant interest in thebiological sciences and medicine.

In concept, solid phase synthesis is relatively simple andstraightforward. An amino acid is attached to a solid phase particle bya linking group on the acid side, and to a protecting group on the amineside. The protecting group is removed so that the second acid (and inparticular it's acid group) can be coupled to the amine group on theoriginal acid. The second (and succeeding) acids are also initiallyprotected, and thus the general sequence is to deprotect, couple, andrepeat until the desired peptide is completed, following which thecompleted peptide is cleaved from the solid phase resin.

Solid phase peptide synthesis had its genesis in 1963 when R. B.Merrifield published the synthesis of a four-acid chain using a solidphase method (R. B. MERRIFIELD ; Solid Phase Peptide Synthesis. I. TheSynthesis of a Tetrapeptide; J. Am. Chem. Soc., 1963, 85 (14), pp2149-2154).

At the time, it was generally recognized that organic reactions could becarried out in this manner, but it was assumed that the Merrifieldmethod would be difficult to adapt to longer peptide sequences in anyrealistic purity. Specifically, Merrifield's suggestion that theisolation steps between and among coupling and deprotection steps couldbe carried out merely by washing and without identification ofintermediates, was considered unlikely to offer long-term success. Inpeptide synthesis, two problems are characteristic: (1) the synthesis ofunwanted byproducts; and (2) the synthesis of some fraction of anundesired sequence based on the presence of unremoved acid from aprevious step or cycle. In particular, a residue of the recently added(“activated”) acid tends to remain after the coupling step and mustaccordingly be removed in some fashion.

Nevertheless, as summarized by CHAN AND WHITE , Fmoc Solid Phase PeptideSynthesis (Oxford University Press 2000), the washing steps provideacceptable purity and the general simplicity of those washing steps andof avoiding detailed characterization of intermediates gives the SPPSmethod its speed and efficiency advantages (e.g., page 1).

Accordingly, as generally well understood in the art, the SPPSdeprotection step is typically carried out by adding an organic base tothe protected acid, then draining the reaction vessel—one of theadvantages of SPPS is that the organic compounds can be handled as ifthey were solids—then washing the deprotected chain. In mostcircumstances, a wash repeated five times is both typical andsatisfactory to remove anything that might create different sequences orundesired byproducts. The coupling step is then carried out followed byanother draining step, and another repetitive wash, with five washesagain being typical.

More recently (e.g., US 20120041173; the contents of which areincorporated entirely herein by reference), it has become recognizedthat adding the deprotecting base for the next cycle will scavenge theactivated acid remaining from the previous cycle, thus reducing oreliminating the number of washing cycles necessary to ensure purity andavoid unwanted sequences.

To interject with a point well understood in this art, improving,accelerating or eliminating any of the SPPS steps becomes geometricallyadvantageous as longer peptide sequences are synthesized. In thisregard, microwave assisted techniques have become widely accepted in theart, following their introduction about a decade ago (e.g., commonlyassigned U.S. Pat. No. 7,393,920, the contents of which are likewiseincorporated entirely herein by reference). Microwave techniques havereduced cycle times from hours to minutes, thus providing multipleadvantages in SPPS and in research or commerce that depends upon SPPS.

To the extent that a newer technique such as microwave assisted solidphase peptide synthesis can be called typical or conventional, the stepof adding the deprotecting base is usually carried out by adding asufficient volume of relatively low concentration that will cover thedrained resin in the reaction vessel and the attached peptide after thecoupling step to ensure that both the scavenging and deprotectionreactions take place.

Doing so, however, creates a thermal slow down (so to speak) in that thevolume of dilute organic base solution is added at room temperature(e.g., 25°) while the coupling step has just been carried out at anelevated temperature, of which temperatures of about 90° C. areexemplary (although not limiting). As expected in a normal heat transfersituation, this reduces the overall temperature of the components in thevessel, which then must be reheated to reach the reaction temperaturerequired for the next deprotection and coupling cycle.

Although these characteristics are disadvantageous only in the strictestsense, an overall advantage always exists when steps in the SPPS cycleare enhanced, accelerated, or simply rendered unnecessary. Suchimprovements become more and more advantageous (and conventional methodsbecome more disadvantageous) as the peptide chain length increases.Thus, speed advantages that might remain proportionally meaningless inconventional organic solid phase reactions (i.e., those that requireonly a few, and perhaps only a single solid phase step) becomeincreasingly important when peptides containing 10, 20, or more acidsare synthesized using SPPS.

SUMMARY

In one aspect the invention is a method of deprotection in solid phasepeptide synthesis in which the improvement comprises adding thedeprotecting composition in high concentration and small volume to themixture of the coupling solution, the growing peptide chain, and anyexcess activated acid from the preceding coupling cycle, and without anydraining step between the coupling step of the previous cycle and theaddition of the deprotection composition for the successive cycle.

In another aspect the invention is a method of deprotection in solidphase peptide synthesis in which the improvement comprises deprotectinga protected amino acid by combining the protected amino acid and aliquid organic base in a reaction vessel and during or after thedeprotection step reducing the ambient pressure in the vessel with avacuum pull to remove the liquid organic base without any intermediatedraining step.

In another aspect the invention is a method of deprotection in solidphase peptide synthesis (SPPS) in which the improvement comprisesdeprotecting a protected amino acid at a temperature of at least about60° C. while providing a path for evaporating base to leave the reactionvessel

In another aspect the invention is a system for microwave assisted solidphase peptide synthesis. In this aspect, the system includes a microwavesource positioned to direct microwave radiation into a microwave cavity,a microwave transparent reaction vessel in the cavity, and a vacuumsource connected to the reaction vessel.

In another aspect the invention is a method of deprotection in solidphase peptide synthesis in which the improvement comprises adding thedeprotecting composition in high concentration and small volume to themixture of the coupling solution, the growing peptide chain, and anyexcess activated acid from the preceding coupling cycle, and without anydraining step between the coupling step of the previous cycle and theaddition of the deprotection composition for the successive cycle, andthereafter reducing the ambient pressure in the vessel with a vacuumpull to remove the deprotecting composition without any draining step.

In another aspect the invention is a method of deprotection in solidphase peptide synthesis which includes the steps of adding thedeprotection composition in high concentration and small volume to themixture of the coupling solution, the growing peptide chain, and anyexcess activated amino acid from the preceding coupling cycle; andwithout any draining step between the coupling step of the previouscycle and the addition of the deprotection composition for thesuccessive cycle which removes at least 50% of the volume of theprevious cycle coupling solution; and with the coupling solution atleast 30° C.

The foregoing and other objects and advantages of the invention and themanner in which the same are accomplished will become clearer based onthe followed detailed description taken in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the conventional steps of SPPSsynthesis.

FIG. 2 is a schematic diagram of an improved version of conventionalSPPS peptide synthesis.

FIG. 3 is a schematic diagram of a first embodiment of the presentinvention.

FIG. 4 is a diagram illustrating the thermal advantages of the currentinvention.

FIG. 5 is a schematic diagram of a second embodiment of the invention.

FIG. 6 is a schematic diagram of an instrument used to carry out themethod of the present invention.

FIG. 7 is a second schematic diagram of portions of the instrument usedto carry out the present invention.

DETAILED DESCRIPTION

FIG. 1 is a schematic diagram of a conventional cycle repeated duringsolid phase peptide synthesis and broadly designated at 20. As set forththerein, the next acid to be added 21 is added in protected fashion to areaction vessel schematically diagramed at 22. The deprotection step 23is carried out in the vessel 22 by adding an organic base in aconcentration of about 20% by volume in dimethyl formamide (DMF). Usefulorganic bases include, but are not limited to piperidine (C₅H₁₁N; CAS No110-89-4), pyrrolidine (C₄H₉N; CAS No 123-75-1), and 4-methyl piperidine(C₆H₁₃N; CAS No. 626-58-4). As indicated by the position of the relativearrows, the deprotection solution 28 is added in advance of the nextacid.

The deprotection solution is then drained (step 24) following which awashing liquid (e.g., methanol or isopropanol) is added to the vesselfor a washing step 25 carried out repetitively with five repetitionsbeing typical. The washing solution is then removed in a second drainingstep 26 which allows the coupling step 27 to take place. The couplingcomposition is then removed in a third draining step 30 followed by asecond washing step 31, again repeated five times.

It will be understood that FIG. 1 is schematic, and that there are manydetails about one SPPS cycle that could be added, but that FIG. 1illustrates the concept sufficiently for the skilled person tounderstand both it and the present invention. In particular, the skilledperson already recognizes that FIG. 1 represents a cycle that is neitherthe step of linking the resin to the first acid, nor does it illustratecleaving a finished peptide from the resin.

FIG. 2 illustrates the improved conventional method referred to in theBackground and broadly designated at 32. In particular, the last washingstep 31 can be omitted because any excess acid left after the couplingstep 27 will be quenched by the deprotection solution (base) added atthe start of the next cycle. Obviously, this requires that deprotectionsolution be added before the next acid 21 is added to the vessel 22.

FIG. 3 illustrates a first embodiment of the invention in which theimprovement comprises adding the deprotection composition in a highconcentration and small volume to the mixture of the coupling solution,the growing peptide chain, and any excess activated acid from thepreceding coupling cycle, and doing so without any draining step betweenthe coupling step of the previous cycle and the addition of thedeprotection composition for the successive cycle.

The use of a small volume in high concentration saves physical space(only a small bottle is needed), avoids the need to prepare a solution,and saves solvent. The method additionally offers a thermal advantage(FIG. 3).

In exemplary versions of the claimed invention, an organic base is usedas the deprotecting composition with piperidine or pyrrolidone or4-methylpiperidine being typical (although not necessarily exclusive)for this purpose. It will be understood, of course, that additionalorganic bases that provide the deprotection function without otherwiseinterfering with the other steps in the method, the growing peptidechain, or the instrument, will be appropriate as well.

In the most exemplary embodiment, the piperidine or pyrrolidine or4-methylpiperidine can be added neat; i.e. as an organic liquid and notin solution. In other circumstances, the piperidine or pyrrolidine or4-methylpiperidine can be added as a highly concentrated solution of atleast about 50% organic base by volume, typically in DMF.

As a further advantage, the high concentration allows the organic baseto be added in a proportionally small volume with a ratio of betweenabout 1:20 and 1:3 being appropriate based upon the volume of thecoupling solution. Piperidine or pyrrolidone or 4-methylpiperidine canbe added in the volume ratio of about 1:5 based upon the volume of thecoupling solution when added neat. In such circumstances, the smallvolume of the deprotecting solution is typically less than 2 ml, andoften less than one milliliter. In exemplary circumstances, betweenabout 0.4 and 1.0 ml of piperidine are added to between about 3.8 and4.2 ml of the mixture of the coupling solution, the growing peptidechain and any excess activated acid.

Expressing the proportion as a percentage, the small volume of thedeprotecting solution is 20% or less of the volume of the mixture of thecoupling solution, the growing peptide chain, and any excess activatedacid.

FIG. 4 illustrates the thermal advantage offered by the invention whichprovides an additional time advantage in each SPPS cycle. As FIG. 4demonstrates, if the coupling step is carried out at temperatures ofabout 90° C., the conventional use of a room temperature (e.g. 25° C.)wash will have the expected thermal effect of lowering the temperatureof peptide and the resin in the vessel in accordance with wellunderstood and relatively simple relationships (e.g., the drop intemperature will be directly proportional to the mass of the addedcooler liquid). Thus, when a washing or draining step is carried outafter coupling, there will be some time interval required to bring thereacting compositions back up to the 90° coupling temperature.

In the invention, however, the addition of a small volume (mass) ofconcentrated base will greatly moderate the degree to which thetemperature drops, thus making it easier and faster to return thecompositions to the required coupling temperatures. In FIG. 4, theconventional thermal profile is indicated by the solid line 34 and thethermal profile provided by the invention is indicated by the dottedline 35. It will be understood, of course, that FIG. 4 is schematic, notdrawn to scale, and illustrative rather than a precise track of anyparticular mixture.

FIG. 5 illustrates another aspect of the invention in which theimprovement comprises deprotecting a protected amino acid by combiningthe protected amino acid and liquid organic base in a reaction vessel,and then during or after the deprotection step reducing the ambientpressure in the vessel to below atmospheric pressure with a vacuum pullto remove the liquid organic base without any intervening draining step.

In general, and as can be confirmed by appropriate resources, theboiling point of piperidine is approximately 106° C. and that of DMF isabout 153° C. As a result the vapor pressure of piperidine will behigher than the vapor pressure of DMF at any given temperature.Accordingly it has now been discovered that pulling a moderate vacuumfrom the vessel can selectively remove the piperidine and completelyavoid the draining step. FIG. 5 illustrates this schematically byshowing the deprotection step 23 followed by an evaporation step 36followed by the draining step (of liquids other than the organic base)and then the coupling step 27. The boiling point of 4-methylpiperidineis 123° C., offering similar advantages.

Expressed alternatively, piperidine's vapor pressure is about 4 mm Hg at25° C., about 39 mm Hg at 50° C., and about 55 mm Hg at 60° C. Forpyrrolidine, the vapor pressure is about 8.4 mm Hg at 25° C. and about102 mm Hg at 60° C. Thus, raising the temperature to 60° C. greatlyencourages the desired evaporation.

Consistent with well understood principles of liquid and vapor pressure,the method can further comprise accelerating the deprotection step byheating the combined protected amino acid and the liquid organic base inthe vessel 22, and then accelerating the removal step further by pullingthe vacuum 36 while heating the vessel contents. When using a microwaveassisted process as described herein (and elsewhere), the microwaveradiation can be used to both accelerate the deprotection step and toaccelerate the vacuum removal step.

In exemplary methods, the pressure can be reduced to below atmosphericpressure, or, expressed in terms of temperatures, the deprotection stepcan be carried out by heating the compositions to at least about 60° C.,and in some cases to between about 81° C. and 99° C., after which thevessel contents can be heated to between about 90° and 110° toaccelerate the vacuum removal step. Functionally, the vacuum and theapplied microwave power should provide the intended enhanced evaporationwithout otherwise adversely affecting the remaining materials in thevessel or causing problems in subsequent steps in the SPPS cycle.

These two improvements in overall SPPS cycles can, be combined, so thatin another aspect, the improvement includes the steps of adding thedeprotecting composition in high concentration and small volume to themixture of the coupling solution, the growing peptide chain, and anyexcess activated acid from the preceding coupling step, and doing sowithout any intervening draining step between the coupling step of theprevious cycle and the addition of deprotection composition for thesuccessive cycle. Thereafter, the ambient pressure in the vessel isreduced with a vacuum pull to remove the deprotecting compositionwithout any draining step.

Combining both improvements in this manner is illustrated by thedifferences between FIG. 1 and FIG. 5 and can allow the cycle to avoidboth the washing steps and two of the draining steps. As set forth inthe Background, any such advantage in an individual cycle will begeometrically multiplied as a longer peptide chain is synthesized.

FIGS. 6 and 7 are schematic illustrations of selected portions of asystem for carrying out the improvements described herein. Mostbasically, the system includes a microwave source illustrated as thediode 40 positioned to direct microwave radiation into a microwavecavity 41, and with a vacuum source shown as the pump 42 connected tothe reaction vessel 22 in the cavity 41. Although the microwave sourceis illustrated as a diode (an IMPATT diode is exemplary), a magnetron isa similarly acceptable source as is a klystron, each of these itemsbeing well understood in the art by the skilled person and can beselected as desired for purposes of convenience, design, or cost, andwithout undue experimentation.

FIG. 6 also shows that microwave radiation from the source 40 istypically directed through a waveguide 43 which provides support to thecavity 41. The vacuum pump 42 pulls from the vessel 22 along line 44 andusually includes a trap 45 which is otherwise conventional (e.g., a coldtrap using liquid nitrogen) and positioned between the vessel and thevacuum pump 42. In the absence of the trap 45, the vacuum pump needs tobe capable of handling the evaporated base and solvents while stilloperating as intended.

As schematically illustrated in FIG. 6, in exemplary embodiments, thecavity 41 can support a single mode of microwave radiation at themicrowave frequencies produced by the microwave source 40. A temperatureprobe 46 (for which a fiber optic device is exemplary) is positioned toread the temperature of the reaction vessel 22 in the cavity 41. Inconjunction with a processor 47 (which can be either internal orexternal to the overall system), the measured temperature can be used todrive the source and to thus increase, decrease, or otherwise moderatethe microwave radiation into the cavity in the most advantageous manner.

As further schematic details, the microwave source 40 is driven by apower supply broadly designated at 50 which in preferred embodiments canbe the switching power supply (and associated methods) set forth in U.S.Pat. No. 6,288,379, the contents of which are incorporated entirelyherein by reference. The basic circuits between the power supply and thediode 40 are likewise illustrated schematically at 51. Basic circuitryof the type required is well understood by those in the relevant arts,need not be described in detail herein, and can be built and operated bythe skilled person without undue experimentation.

FIG. 7 schematically illustrates a few additional details of the systemfor carrying out the method of the invention. In FIG. 7 the vessel isagain designated at 22, and FIG. 7 further illustrates that the vessel22 includes a frit 52 (typically made of glass) and a spray head 53. Thefrit 52 permits liquids to be drained from the reaction vessel 22 andthe spray head 53 delivers compositions to the reaction vessel 22. Otherequivalent fixtures can be selected by the skilled person without undueexperimentation.

In particular, FIG. 7 illustrates a nitrogen supply 54 which isconnected to a plurality of supply bottles 55 which for schematicpurposes are illustrated as Erlenmeyer flasks. A plurality of meteredloops are schematically illustrated by lines 56, 57, and 58 and connectthe nitrogen supply to the supply bottles 55; and corresponding lines60, 61, and 62 then connect to a common line 63 that reaches the sprayhead 53 for delivery to the vessel 22. A separate line 63 providesnitrogen from the source 54 to the liquids and resin in the vessel 22 toagitate (bubble) the contents of the vessel 22 to carry out appropriatemixing and circulation during deprotection, coupling, and cleavagereactions.

Nitrogen is helpful under these circumstances because it is relativelyinexpensive, widely available, and inert to the reactions being carriedout and to the equipment in the instrument or system. It will thus beunderstood that other inert gases, including the noble gases, can beused for this purpose, but in most cases will simply be more expensiveand less widely available. In a functional sense, any gas that willavoid interfering chemically with the ongoing reactions or with theinstrument will be appropriate.

In a manner consistent with the diagram of FIG. 6, the nitrogen supplyand the metered loop can connect to the processor 47 so that theprocessor 47 can control the manner in which the compositions aredispensed from the vessels 55 to the reaction vessel 52. Although notillustrated, the skilled person will recognize that the simple schematicline connections (64 and 65) are in practice combination of tubes(pipes), valves, and controls for those lines; e.g., in practice line 64represents a connection between a valve or manifold in line 58, acontroller for that line, and the processor 47. The same relationshipshold true for the line 65 between the nitrogen supply 54 and theprocessor 47.

Experimental (Predictive)

Materials and Methods

Reagents

All Fmoc amino acids were obtained from Novabiochem (San Diego, CA) andcontained the following side chain protecting groups: Asn(Trt),Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt), Lys(Boc),Ser(tBu), Thr(tBu), Trp(Boc), and Tyr(tBu).N-[(1H-Benzotriazol-1-yl)(dimethylamino)methylenel-N-methylmethanaminiumhexafluorophosphate Noxide (HBTU), N-hydroxybenzotriazole (HOBO, andbenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate(PyBOP), were also obtained from Novabiochem. Diisopropylethylamine(DIEM, N-methylmorpholine (NMM), collidine (TMP), piperidine,piperazine, trifluoroacetic acid (TFA), thioanisole, 1,2-ethanedithiol(EDT), and phenol were obtained from Sigma Aldrich (St. Louis, Mo.).Dichloromethane (DCM), N,N-Dimethylformamide (DMF), Nmethylpyrrolidone(NMP), anhydrous ethyl ether, acetic acid, HPLC grade water, and HPLCgrade acetonitrile were obtained from VWR (West Chester, Pa.).

SPHERITIDE™resin: Trityl linker was prepared using SPHERITIDE™ resin(CEM Corporation; Matthews, N.C.; USA). The SPHERITIDE™ resin consistsof poly-e-lysine cross-linked with multifunctional carboxylic acids.

CEM LIBERTYTM Automated Microwave Peptide Synthesizer

The LIBERTY™ system (CEM Corporation, Matthews, N.C.) is a sequentialpeptide synthesizer capable of complete automated synthesis includingcleavage of up to 12 different peptides. The LIBERTY™ system uses thesingle-mode microwave reactor, DISCOVER™, which has been widely used inthe organic synthesis industry. The LIBERTY™ synthesizer uses a standard30 milliliter (ml) Teflon® glass fritted reaction vessel for 0.025-1.0millimole (mmol) syntheses. The reaction vessel features a spray headfor delivery of all reagents and a fiber-optic temperature probe forcontrolling the microwave power delivery. The system utilizes up to 25stock solutions for amino acids and seven reagent ports that can performthe following functions: main wash, secondary wash, deprotection,capping, activator, activator base, and cleavage. The system usesnitrogen pressure for transfer of all reagents and to provide an inertenvironment during synthesis. Nitrogen bubbling is used for mixingduring deprotection, coupling, and cleavage reactions. The system usesmetered sample loops for precise delivery of all amino acid, activator,activator base, and cleavage solutions. The LIBERTY™ synthesizer iscontrolled by an external computer, which allows for complete control ofeach step in every cycle.

Peptide Synthesis: VYWTSPFMKLIHEQCNRADG-NH2

A model peptide containing all 20 amino acids was synthesized under avariety of conditions using the CEM LIBERTY™ automated microwave peptidesynthesizer on 0.152 g Spheritide™ resin (0.66 meq/g substitution).Deprotection was performed in two stages using a fresh reagent each timewith (i) 80% piperidine in DMF; or (ii) piperidine neat. In each case,0.8 ml of the piperidine was added to 4.0 ml of the coupling solutionremaining from the addition of the previous acid. An initialdeprotection of 30 s at 50 W (5 min at 0 W for conventional synthesis)was followed by a 3-min deprotection at 50 W (15 min at 0 W forconventional synthesis) with a maximum temperature of 80° C.

No draining step was carried out between the coupling step of a previouscycle and the addition of the piperidine for the successive cycle.

After deprotection, the piperidine was removed by applying a vacuum thatreduced the ambient pressure in the reaction vessel to below atmosphericpressure. Removal was enhanced by applying microwave power at 50 W for 3minutes.

Coupling reactions were performed in the presence of a 5-fold molarexcess of 0.2 M Fmoc-protected amino acids dissolved in DMF with varioustypes of activation: (i) HBTU:DIEA:AA (0.9:2:1); HBTU:HOBt:DIEA:AA(0.9:1:2:1); (iii) PyBOP:DIEA:AA (0.9:2:1); (iv) HBTU:NMM:AA (0.9:2:1);and (v) HBTU:TMP:AA (0.9:2:1), double coupling on valine. Couplingreactions were for 5 min at 40 W (30 min at 0 W for conventionalsynthesis) with a maximum temperature of 80° C. In later experiments,coupling conditions of cysteine and histidine were altered to 2 min at 0W followed by 4 min at 40 W with a maximum temperature of 50° C.Cleavage was performed using 10 ml of Reagent K(TFA/phenol/water/thioanisole/EDT; 82.5/5/5/5/2.5) for 180 min.Following cleavage, peptides were precipitated out and washed usingice-cold anhydrous ethyl ether.

Peptide Analysis

Prior to LC-MS analysis, all peptides were dissolved in 10% acetic acidsolution and lyophilized to dryness. Analytical HPLC of peptide productswas performed using a Waters Atlantis dC18 column (3 μm, 2.1×100 mm) at214 nm. Separation was achieved by gradient elution of 5-60% solvent B(solvent A=0.05% TFA in water; solvent B=0.025% TFA in acetonitrile)over 60 min at a flow rate of 0.5 ml/min. Mass analysis was performedusing an LCQ Advantage ion trap mass spectrometer with electrosprayionization (Thermo Electron, San Jose, Calif.). Racemization analysis ofamino acids was performed by C.A.T. GmbH & Co. (Tuebingen, Germany)using a published GC-MS method that involves hydrolysis of the peptidein 6 N DCl/D2O (The Peptides: Analysis, Synthesis, Biology, ERHARD GROSSeditor).

In another embodiment, the invention presents a novel process wherebythe coupling and deprotection steps occur within the same solvent. Inthis process concentrated base is added directly to the resin couplingsolution after a desired period of time for the coupling to occur. Thedeprotection step is then immediately started when the base is added.Therefore, the onset of the deprotection step is immediately after thecoupling step without any time delay. Additionally, only a small volumeof base is required since it can use the solvent present from thecoupling reaction. This requires a sophisticated reagent delivery systemfor the base that is accurate at very small volumes (0.5 mL) with rapiddelivery. Typically, a 20% solution of base (piperidine) in solvent isused for the deprotection step. Excess base concentration can increasebase-catalyzed side reactions and therefore significant solvent isrequired. This means that significant solvent can be saved from thisprocess by adding concentrated base to the coupling solvent.

To demonstrate the effectiveness of this new process a batch of 24peptides were assembled using an automated peptide synthesizer modifiedto perform the in-situ solvent recycling step during each cycle.

Materials and Methods

All peptides were synthesized using a Liberty Blue PRIME system (CEMCorporation; Matthews, N.C.; USA) allowing for automated in-situ solventrecycling and evaporation based washing. The peptides were synthesizedat 0.05mmol scale with 10 equivalents of amino acid using CarboMAX™coupling with AA/DIC/Oxyma (1:2:1) based activation for 100 sec at 90°C. ProTide resins (CEM Corporation; Matthews, N.C.; USA) based onTentaGel® technology were used for synthesis with either a Rink Amidelinker or a Cl-TCP(Cl) linker with unactivated loading of the firstamino acid with DIEA at 90° C. for 5 min. The deprotection step wasperformed for 50 sec at 95° C. and initiated by adding 0.5 mL of 50%pyrrolidine directly to the coupling solution. A single 1×4 mL wash wasused in between the deprotection and coupling steps. Peptides werecleaved with TFA/TIS/H2O/DODt (92.5:2.5:2.5:2.5) for 30 min at 38° C.using a RAZOR cleavage system (CEM Corporation; Matthews, N.C.; USA).

Results and Discussion:

All peptides synthesized in Table 1 gave the desired target as the majorpeak with a standard cycle time of 2 minutes and 58 seconds. The in-situsolvent recycling process allowed for 0.5 mL of a concentratedpyrrolidine (BP 87° C.) solution to be added to the end of the couplingstep (without draining) An advantage of this setup was that thedeprotection immediately proceeded very close to the desired temperature(95° C.) since the coupling solution was already at 90° C. During thedeprotection process a vacuum was applied and the pyrrolidine wasevaporated and subsequently condensed in the waste container. Thisallowed only a single wash step (1×4 mL) to be required at the end ofthe deprotection step.

TABLE 1 Automated Sequential Batch Synthesis of 24 Peptides UPLCSynthesis # Peptide Disease Area Resin Used Purity (%) Time 1 GRPRegulates Gastrin RA ProTide 81 1:22 VPLPAGGGTVLTKMYPRGNHWAVGHLM-NH ₂Release 2 Glucagon Hypoglycemia RA ProTide 75 1:28 H-HSQGTFTSDYSKYLDSRRAQDFVQWLMNT- NH2 3 Bivalirudin Blood thinnerC1-2-C1-Trt 71 1:05 H-fPRPGGGGNGDFEEIPEEYL-OH 4 AngiotensinVasoconstrictor C1-2-C1-Trt 82 0:30 H-NRVYVHPF-OH 5 PTH 1-34Osteoporosis RA ProTide 70 1:43 H- SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ 6 Gonadorelin Fertility RA ProTide 91 0:35 pEHWSYGLRPG-NH₂ 7Triptorelin Breast Cancer, RA ProTide 73 0:35 pEHWSYwLRPG-NH₂Prostrate Cancer, Fertility 8 Liraglutide Diabetes RA ProTide 80 1:31H-HAEGTFTSDVSSYLEGQAAK(γ-E- palmitoyl)EFIAWLVRGRG-NH₂ 9 ExenatideDiabetes RA ProTide 74 1:58 H- HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ 10 MOG (35-55) Multiple Sclerosis RA ProTide 71 1:05H-MEVGWYRSPFSRVVHLYRNGK-NH₂ 11 Secretin Osmoregulation RA ProTide 701:19 H-HDGTFTSELSRLRDSARLQRLLQGLV-NH₂ 12 Teriparatide OsteoporosisRA ProTide 60 1:43 H- SVSEIQLMHNLGKHLNSMERVEWLRKKLQD VHNF-NH₂ 13GLP-1 (7-37) Diabetes RA ProTide 74 1:34 H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR G-NH₂ 14 Magainin 1 Antibiotic RA ProTide79 1:11 H-GIGKFLHSAGKFGKAFVGEIMKS-NH₂ 15 Tetracosactide Adrenal CortexRA ProTide 77 1:13 H-SYSMEHFRWGKPVGKKRRPVKVYP-NH₂ stimulant 16[Arg8]-Vasopressin Hormone (blood RA ProTide 94 0:32 H-CYFQNCPRG-NH₂vessel contraction) 17 Ubiquitin Protein signaling RA ProTide ≥60 3:44MQIFVKTLTGKTITLEVEPSDTIENVKAKIQD agent KEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-NH₂ 18 Parasin I Antibiotic RA ProTide 87 0:59H-KGRGKQGGKVRAKAKTRSS-NH₂ 19 Dynorphin A Opioid Research RA ProTide 710:53 H-YGGFLRRIRPKLKWDNQ-NH₂ 20 ACP Fatty Acid RA ProTide 92 0:32H-VQAAIDYING-NH₂ Synthesis 21 BAM 3200 Opioid Research RA ProTide 701:16 H-YGGFMRRVGRPEWWMDYQKRYGGFL- NH₂ 22 HIV-TAT (47-57) HIV/AIDSRA ProTide 93 0:31 Fmoc-YGRKKRRQRRR-NH₂ Research 23 HIV-TAT (48-60)HIV/AIDS RA ProTide 88 0:39 Fmoc-GRKKRRQRRRPPQ-NH₂ Research 24Pramlintide Diabetes RA ProTide 72 1:52 KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY--NH₂

Total Synthesis Time for Entire Batch: 32.6 Hours

This new process provided a significant reduction in standard cycle time(2 minutes 57 seconds) from (a)—elimination of the coupling drain time,(b)—elimination of the deprotection delivery time between steps, and(c)—elimination of the temperature ramp time for the deprotection stepthereby allowing a shorter deprotection time to be used. Additionally,significant solvent savings were possible with the complete eliminationof the deprotection solvent during each cycle.

In the drawings and specification there has been set forth a preferredembodiment of the invention, and although specific terms have beenemployed, they are used in a generic and descriptive sense only and notfor purposes of limitation, the scope of the invention being defined inthe claims.

1. A system for microwave assisted solid phase peptide synthesiscomprising: a microwave source positioned to direct microwave radiationinto a microwave cavity; a microwave transparent reaction vessel in saidcavity; and a vacuum source connected to said reaction vessel.
 2. Amethod according to claim 1 further comprising a trap between saidreaction vessel and said vacuum Source.
 3. A method according to claim 1wherein said cavity can support a single mode of microwave radiation atthe microwave frequencies produced by said microwave source.
 4. A methodaccording to claim 3 where wherein said reaction vessel comprises: a(glass) frit for draining liquids from said reaction vessel; and a sprayhead for delivery of reagents to said reaction vessel.
 5. A systemaccording to claim 1 further comprising a fiber optic temperature probepositioned to read the temperature of said reaction vessel in saidcavity (for controlling the microwave power delivered to said reactionvessel).
 6. A system according to claim 4 that incorporates nitrogenpressure to transfer all reagents and to provide an inert environmentduring peptide synthesis.
 7. A system according to claim 6 furthercomprising a nitrogen source in communication with said reaction vesselto bubble the contents of said reaction vessel for mixing duringdeprotection, coupling, and cleavage reactions.
 8. A system according toclaim 1 further comprising a processor for controlling every step inevery SPPS cycle carried out in said system.